Characterization of Mouse ACTH in Plasma and in Extracts of Pituitary and of Adrenotropic Pituitary Tumor RAFAEL COSLOVSKY,1 BRUCE SCHNEIDER, AND ROSALYN S. YALOW23 Solomon A. Berson Research Laboratory, Veterans Administration Hospital, Bronx, New York 10468 [125I]hACTH (little ACTH). Big and intermediate ACTH are urea-stable. Controlled tryptic digestion of mouse-tumor big ACTH results within 10 seconds in conversion to an intermediate component followed by continued loss of immunoreactivity. Under the same conditions of tryptic digestion of intermediate ACTH, there is only continuous loss of immunoreactivity with no change of hormonal form. These findings strengthen the hypothesis that mouse intermediate ACTH is not a precursor for little ACTH. (Endocrinology 97: 1308, 1975)
ABSTRACT. The predominant component of immunoreactive ACTH in the plasma of adrenalectomized normal mice and of mice bearing the adrenotropic mouse pituitary tumor, AtT-20, and in extracts of the normal mouse pituitary and pituitary tumor, has an elution volume on Sephadex G-50 gel filtration approximately midway between the void volume and the elution volume of human ACTH (1-39 peptide). The tumor extracts are shown to contain, in addition to this intermediate ACTH, 2 other components of immunoreactive ACTH, one which coelutes with I3I Ilabeled albumin (big ACTH) and the other with
T
HE ACTH content of transplanted adrenotropic mouse pituitary tumor, AtT-20 (1,2), was first characterized by Canfield et al. (3) who found only a single form of ACTH which had an amino acid composition and a molecular weight similar to that of porcine ACTH (4500 dalton mol wt). Subsequently Orth and associates (4) detected another form of ACTH in similar tumor extracts as well as in the pituitaries of normal LAFj mice, in cultured AtT-20 cells, and in the medium in which the cells were cultured. On the basis of Sephadex gel filtration they estimated this new form of ACTH to have a molecular weight between 6000 and 7800 daltons and demonstrated that it has adrenotropic activity. We have confirmed that this new hormonal form, which we called intermediate ACTH, is in the mouse pituitary and have found it also in pituitaries of the rat, rabbit, hamster, and cow (5). In the present report we demonstrate that the intermediate ACTH is also found in the Received March 6, 1975. 1 Fellow of the Solomon A. Berson Fund for Medical Research. 2 V.A. Project no. 9678-01, Mt. Sinai School of Medicine, CUNY. 3 Address reprint requests to: Dr. Yalow, Solomon A. Berson Research Laboratory, Veterans Administration Hospital, Bronx, New York 10468.
plasma of tumor-bearing or adrenalectomized mice, and we further characterize the nature of the intermediate ACTH. We have also detected a "big" ACTH in the tumor extract that is convertible by controlled tryptic digestion to an intermediatelike ACTH whereas intermediate ACTH is not demonstrably converted to a smaller immunoreactive form under the same conditions. Materials and Methods Adrenalectomized and non-adrenalectomized LAF, mice received transplants of AtT-20 pituitary tumors. Tumors were harvested when their average diameter was 1-2 cm and were kept frozen until extracted. Plasma was obtained from non-tumor-bearing adrenalectomized and from tumor-bearing LAF, mice by exsanguination or tapping the jugular vein under Nembutal anesthesia. Normal pituitaries from Swiss-Webster mice were obtained from Pel-Freeze Biologicals, Inc., Rogers, Arkansas. Tumors and pituitaries were weighed, minced while still frozen, and extracted in boiling water as previously described (6,7). Plasma samples were not treated before radioimmunoassay. To determine the form of immunoreactive ACTH in the tissue extracts or plasma, portions were fractionated on Sephadex G-50 fine columns by methods previously described (6-8), except that the columns were equilibrated with and eluted with 0.1M NaCl containing 0.5% 2-mer-
1308
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CHARACTERIZATION OF MOUSE ACTH captoethanol, 0.025% sodium azide, and 0.25% human serum albumin (HSA). Adsorption of ACTH to glassware did not occur when HSA was used instead of the 10% hormone-free human plasma previously employed. The stability of the larger forms of ACTH in urea was evaluated by fortifying portions of the pooled Sephadex gel eluates obtained from the "big" and "intermediate" regions with 8 M urea, incubating for 12 h at 4 C and refractionating on a G-50 column equilibrated with and eluted with die column diluent fortified with 8 M urea. Sephadex gel eluates obtained from the "big" and "intermediate" regions were fortified with phosphate buffer and treated with chymotrypsinfree trypsin at a final concentration of 40 (xg trypsin/ml as previously described (6,9). Radioimmunoassay of ACTH was performed with some modification of methods previously employed (6,7,10,11). Purified human ACTH (Upton-Lerner 8B) and synthetic human ACTH, both supplied by the National Pituitary Agency, were used interchangeably for labeling and as standards. The standard diluent for the assay was 0.02 M barbital buffer containing 0.5% 2-mercaptoethanol and 0.6% human serum albumin. In some experiments the diluent also included 1% normal guinea pig serum. No differences were noted in assays when the guinea pig serum was not employed and occasional lots of commercial guinea pig serum appeared to damage the labeled ACTH. Tissue extracts were assayed at dilutions of 1:100 or greater in the standard diluent. Plasma samples were assayed at a dilution of 1:25 or greater. Standards for the assays were prepared in the same diluent (hormone-free plasma, standard diluent, or Sephadex gel diluent) and in the same volumes as used for die samples which were assayed. All concentrations are expressed as mass content per unit volume immunoehemiTABLE 1. Immunoreactive ACTH in tissue extracts
Tissue Normal mouse pituitary Tumor tissue from LAF, mice Non-adrenalectomized #708 #910 Adrenalectomized #911 #716
ACTH concentration 103 3.5 55 63 167
1309
SEPHADEX G 5 0 FINE GEL FILTRATION TUMOR EXTRACT ICOCh
88 /jg/g
WET TISSUE
to UJ
CPM
100
_l UJ
A
_l UJ
REFRACTIONATION OF BIG
X UJ Q Q. UJ CO
REFRACTIONATION OF INTERMEDIATEl
120
UJ O
O
o o
REFRACTIONATION OF LITTLE I
60 -\
0 PERCENT I3I
20 40 60 80 100 OF ELUTION VOLUME BETWEEN
I-ALBUMIN AND
l31
1"
FlG. 1. Sephadex G50 gelfiltrationof extract of mouse pituitary tumor and refractionation of "big," "intermediate," and "little" components. Radioiodinelabeled marker molecules were added to samples before application to the column to locate the elution region of albumin, human ACTH, and iodide. cally equivalent to the purified natural or synthetic human ACTH used as standard. Assays were generally performed with our guinea pig anti-porcine ACTH serum (330-5-27) which does not distinguish between equimolar amounts of big or little (1-39) human ACTH (6,7,9). Hormonal fragments substantially smaller than the 1-39 peptide react poorly with this antiserum (6,10). The relative immunologic potencies of the various hormonal forms of mouse ACTH as measured with our antiserum were compared with the potencies as measured with a rabbit anti-porcine
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COSLOVSKY, SCHNEIDER AND YALOW
1310
ACTH serum prepared by Dr. Charles D. West, University of Utah Medical Center, and supplied to us through the National Pituitary Agency. Results The concentrations of immunoreactive ACTH in extracts of the pooled pituitaries from normal mice, from tumors obtained from the 2 adrenalectomized mice, and from the 2 intact tumor-bearing mice are given in Table I. The ACTH concentrations in the tumors from the 2 adrenalectomized mice averaged about that in the pooled pituitaries of normal mice. In extracts of the tumor from the 2 non-adrenalectomized mice, the ACTH concentrations averaged somewhat lower than those of normal mice pituitaries.
lindo • 1975 Vol 97 • No 5
Since only 2 tumors of each type were studied, these differences may not be significant. The predominant component (intermediate ACTH) of immunoreactivity in all tumor extracts had an elution volume on Sephadex G-50 gel filtration about midway between the void volume, as marked by 131I-albumin, and the elution volume of the 1-39 peptide, as marked by 125I-human ACTH (Fig. 1). There were 2 other peaks of immunoreactive ACTH, one in the void volume (big ACTH) and one in the same region as the labeled human ACTH (little ACTH). Each component maintained its integrity on refractionation. The pattern observed was similar to that previously found on fractionation of the normal mouse pituitary (5).
SEPHADEX G 5 0 FINE GEL FILTRATION [BIG""ACTH|
UNTREATED
UREA TREATED
"3001
601 Q. i
(f)
CPM
LU I3I
§
I-ALB
'"I-ACTH
A
U
i
i
i
I3l r
i
-100
A i
i
4001 Q:
frNTERMEDiATEl J ACTH i"
60i
lxl O
O O X O
ABSTRACT. The predominant component of immunoreactive ACTH in the plasma of adrenalectomized normal mice and of mice bearing the adrenotropic mouse pituitary tumor, AtT-20, and in extracts of the normal mouse pituitary and pituitary tumor, has an elution volume on Sephadex G-50 gel filtration approximately midway between the void volume and the elution volume of human ACTH (1-39 peptide). The tumor extracts are shown to contain, in addition to this intermediate ACTH, 2 other components of immunoreactive ACTH, one which coelutes with I3I Ilabeled albumin (big ACTH) and the other with
T
HE ACTH content of transplanted adrenotropic mouse pituitary tumor, AtT-20 (1,2), was first characterized by Canfield et al. (3) who found only a single form of ACTH which had an amino acid composition and a molecular weight similar to that of porcine ACTH (4500 dalton mol wt). Subsequently Orth and associates (4) detected another form of ACTH in similar tumor extracts as well as in the pituitaries of normal LAFj mice, in cultured AtT-20 cells, and in the medium in which the cells were cultured. On the basis of Sephadex gel filtration they estimated this new form of ACTH to have a molecular weight between 6000 and 7800 daltons and demonstrated that it has adrenotropic activity. We have confirmed that this new hormonal form, which we called intermediate ACTH, is in the mouse pituitary and have found it also in pituitaries of the rat, rabbit, hamster, and cow (5). In the present report we demonstrate that the intermediate ACTH is also found in the Received March 6, 1975. 1 Fellow of the Solomon A. Berson Fund for Medical Research. 2 V.A. Project no. 9678-01, Mt. Sinai School of Medicine, CUNY. 3 Address reprint requests to: Dr. Yalow, Solomon A. Berson Research Laboratory, Veterans Administration Hospital, Bronx, New York 10468.
plasma of tumor-bearing or adrenalectomized mice, and we further characterize the nature of the intermediate ACTH. We have also detected a "big" ACTH in the tumor extract that is convertible by controlled tryptic digestion to an intermediatelike ACTH whereas intermediate ACTH is not demonstrably converted to a smaller immunoreactive form under the same conditions. Materials and Methods Adrenalectomized and non-adrenalectomized LAF, mice received transplants of AtT-20 pituitary tumors. Tumors were harvested when their average diameter was 1-2 cm and were kept frozen until extracted. Plasma was obtained from non-tumor-bearing adrenalectomized and from tumor-bearing LAF, mice by exsanguination or tapping the jugular vein under Nembutal anesthesia. Normal pituitaries from Swiss-Webster mice were obtained from Pel-Freeze Biologicals, Inc., Rogers, Arkansas. Tumors and pituitaries were weighed, minced while still frozen, and extracted in boiling water as previously described (6,7). Plasma samples were not treated before radioimmunoassay. To determine the form of immunoreactive ACTH in the tissue extracts or plasma, portions were fractionated on Sephadex G-50 fine columns by methods previously described (6-8), except that the columns were equilibrated with and eluted with 0.1M NaCl containing 0.5% 2-mer-
1308
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CHARACTERIZATION OF MOUSE ACTH captoethanol, 0.025% sodium azide, and 0.25% human serum albumin (HSA). Adsorption of ACTH to glassware did not occur when HSA was used instead of the 10% hormone-free human plasma previously employed. The stability of the larger forms of ACTH in urea was evaluated by fortifying portions of the pooled Sephadex gel eluates obtained from the "big" and "intermediate" regions with 8 M urea, incubating for 12 h at 4 C and refractionating on a G-50 column equilibrated with and eluted with die column diluent fortified with 8 M urea. Sephadex gel eluates obtained from the "big" and "intermediate" regions were fortified with phosphate buffer and treated with chymotrypsinfree trypsin at a final concentration of 40 (xg trypsin/ml as previously described (6,9). Radioimmunoassay of ACTH was performed with some modification of methods previously employed (6,7,10,11). Purified human ACTH (Upton-Lerner 8B) and synthetic human ACTH, both supplied by the National Pituitary Agency, were used interchangeably for labeling and as standards. The standard diluent for the assay was 0.02 M barbital buffer containing 0.5% 2-mercaptoethanol and 0.6% human serum albumin. In some experiments the diluent also included 1% normal guinea pig serum. No differences were noted in assays when the guinea pig serum was not employed and occasional lots of commercial guinea pig serum appeared to damage the labeled ACTH. Tissue extracts were assayed at dilutions of 1:100 or greater in the standard diluent. Plasma samples were assayed at a dilution of 1:25 or greater. Standards for the assays were prepared in the same diluent (hormone-free plasma, standard diluent, or Sephadex gel diluent) and in the same volumes as used for die samples which were assayed. All concentrations are expressed as mass content per unit volume immunoehemiTABLE 1. Immunoreactive ACTH in tissue extracts
Tissue Normal mouse pituitary Tumor tissue from LAF, mice Non-adrenalectomized #708 #910 Adrenalectomized #911 #716
ACTH concentration 103 3.5 55 63 167
1309
SEPHADEX G 5 0 FINE GEL FILTRATION TUMOR EXTRACT ICOCh
88 /jg/g
WET TISSUE
to UJ
CPM
100
_l UJ
A
_l UJ
REFRACTIONATION OF BIG
X UJ Q Q. UJ CO
REFRACTIONATION OF INTERMEDIATEl
120
UJ O
O
o o
REFRACTIONATION OF LITTLE I
60 -\
0 PERCENT I3I
20 40 60 80 100 OF ELUTION VOLUME BETWEEN
I-ALBUMIN AND
l31
1"
FlG. 1. Sephadex G50 gelfiltrationof extract of mouse pituitary tumor and refractionation of "big," "intermediate," and "little" components. Radioiodinelabeled marker molecules were added to samples before application to the column to locate the elution region of albumin, human ACTH, and iodide. cally equivalent to the purified natural or synthetic human ACTH used as standard. Assays were generally performed with our guinea pig anti-porcine ACTH serum (330-5-27) which does not distinguish between equimolar amounts of big or little (1-39) human ACTH (6,7,9). Hormonal fragments substantially smaller than the 1-39 peptide react poorly with this antiserum (6,10). The relative immunologic potencies of the various hormonal forms of mouse ACTH as measured with our antiserum were compared with the potencies as measured with a rabbit anti-porcine
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COSLOVSKY, SCHNEIDER AND YALOW
1310
ACTH serum prepared by Dr. Charles D. West, University of Utah Medical Center, and supplied to us through the National Pituitary Agency. Results The concentrations of immunoreactive ACTH in extracts of the pooled pituitaries from normal mice, from tumors obtained from the 2 adrenalectomized mice, and from the 2 intact tumor-bearing mice are given in Table I. The ACTH concentrations in the tumors from the 2 adrenalectomized mice averaged about that in the pooled pituitaries of normal mice. In extracts of the tumor from the 2 non-adrenalectomized mice, the ACTH concentrations averaged somewhat lower than those of normal mice pituitaries.
lindo • 1975 Vol 97 • No 5
Since only 2 tumors of each type were studied, these differences may not be significant. The predominant component (intermediate ACTH) of immunoreactivity in all tumor extracts had an elution volume on Sephadex G-50 gel filtration about midway between the void volume, as marked by 131I-albumin, and the elution volume of the 1-39 peptide, as marked by 125I-human ACTH (Fig. 1). There were 2 other peaks of immunoreactive ACTH, one in the void volume (big ACTH) and one in the same region as the labeled human ACTH (little ACTH). Each component maintained its integrity on refractionation. The pattern observed was similar to that previously found on fractionation of the normal mouse pituitary (5).
SEPHADEX G 5 0 FINE GEL FILTRATION [BIG""ACTH|
UNTREATED
UREA TREATED
"3001
601 Q. i
(f)
CPM
LU I3I
§
I-ALB
'"I-ACTH
A
U
i
i
i
I3l r
i
-100
A i
i
4001 Q:
frNTERMEDiATEl J ACTH i"
60i
lxl O
O O X O
